The hypothalamo-neurohypophyseal system regulates the osmotic composition of body fluids by adjusting the rate of secretion of antidiuretic hormone to the level of tonicity of the plasma. It is the objective of this proposal to develop new techniques for studying the osmo-regulatory mechanism in organ and cell cultures of the neurosecretory complex. The following systems will be studied in vitro: a) isolated posterior pituitaries attached via an intact pituitary stalk to hypothalamic tissue blocks of different dimensions (adult toads, chick embryos), b) pre-optic hypothalamic nuclei grown alone and in combination with other fragments of the osmo-regulatory complex in tissue culture, c) individual perikarya of the preoptic nucleus grown alone and in combination with other classes of hypothalamic and posterior pituitary cells. The acute and chronic responses of these in vitro preparations to physiological stimuli and to a select group of biological active agents will be analyzed in terms of the ability of the neurosecretory neurons to synthesize, store, and release the nonapeptide, 8-arginine-vasotocin. The rate of vasotocin synthesis and release will be estimated with the toad bladder bio-assay by measuring the hydroosmotic activity of acetic acid extracts of tissue homogenates and by measuring progressive changes in the hydroosmotic activity of the culture medium. It is hoped that the studies proposed will identify the "osmo-receptor cells" and define some of the steps that intervene between activation of these receptors and release of antidiuretic hormone into the general circulation.